Insights from a decade of surveillance: Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Norway from 2008 to 2017

Aim Norway has a low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and reporting of all MRSA cases has been mandatory, including infections and carriage, since 1995 and 2005 accordingly. This provides a unique window to study the spread of MRSA in Norway over time. The aim of this study was to analyze the nationwide trends in the molecular epidemiology of MRSA in Norway over a period of 10 years. Methods Clinical and epidemiological data as well as bacterial genotype (spa-type and PVL) were analyzed for all reported MRSA cases in Norway in the period 2008–2017. Results During the study period, there were 15,200 MRSA cases reported in Norway, from 14,386 patients. The notification rate per 100,000 population increased by 15% annually, rising from 14.2 in 2007 to 48.6 in 2017. This increase was primarily driven by MRSA carriage and community-associated MRSA cases. The incidence of invasive infections remained stable and low, at less than 0.5. The incidence of healthcare-associated MRSA showed an increasing trend, while the number of outbreak-related cases, particularly those associated with nursing homes, decreased. Overall, there were significantly more MRSA infections in males than females. Interestingly, there was a significantly higher prevalence of MRSA infections in female young adolescents compared to males. spa-typing revealed a very heterogeneous MRSA population (D = 0.97), predominantly impacted by international travel and migration patterns, and less by domestic spread in the community. Conclusions This study highlights that Norway, while still classified as a low-prevalence country, has experienced a significant increase in the incidence of MRSA between 2008 and 2017, which can predominantly be attributed to CA-MRSA and MRSA carriage.


Introduction
Staphylococcus aureus is part of the normal bacterial flora in 20-30% of the population, where carriage is typically found in the nose, throat or skin [1].S. aureus is also a major human pathogen causing nosocomial and community-acquired infections, ranging in severity from skin and soft tissue infections to bone-and joint infections, pneumonia, endocarditis and bloodstream infections [2].
S. aureus has a wide array of virulence factors, and the ability to acquire or develop resistance to all classes of clinically relevant antibiotics [3].Presence of the staphylococcal cassette chromosome mec (SCCmec), a genomic island that holds mecA/mecC-genes encoding methicillin resistance, is the defining trait of methicillin-resistant S. aureus (MRSA) [4,5].MRSA are resistant to all beta-lactam antibiotics, including penicillins and cephalosporins, which can lead to delayed, limited options for or failure of treatment [3].Infections due to antibioticresistant bacteria such as MRSA are thus associated with increased morbidity and mortality in healthcare institutions, and the spread of these pathogens is considered a major threat to modern healthcare [6].
Norway has a very low prevalence of MRSA compared to other countries [7], and a "search and destroy" policy in order to prevent MRSA from becoming endemic in healthcare institutions.This involves risk-based screening, contract tracing, isolation of MRSA positive patients and work-restrictions and decolonization treatment of MRSA positive healthcare personnel [8].Furthermore, all new laboratory confirmed cases of MRSA, including both infections and carriage, are reported to the Norwegian Surveillance System for Communicable Diseases (MSIS), and all MRSA strains are confirmed and genotyped at the Norwegian MRSA reference laboratory.This provides a unique window into the spread of MRSA in Norway over time, and the aim of the current study was thus to analyze trends in the molecular epidemiology of MRSA in Norway based on 10 years of surveillance.Specific objectives included describing temporal changes in genotypes during the study period, and detecting potential associations between MRSA genotype and sex, age groups, infection or carriage, country of acquisition and outbreaks in healthcare institutions.

Study design and population
In this study, a case was defined as laboratory-confirmed MRSA notified to MSIS and/or MRSA strains confirmed by the Norwegian MRSA reference laboratory.All cases of MRSA in Norway from 2008 to 2017 were included, limited to the first case per year per individual.

Clinical and epidemiological data
Clinical and epidemiological data on all cases was collected from MSIS (accessed 29.10.2021) and request forms from the referring laboratory or treating physician (accessed through the laboratory information system, LIMS, 22.11.2021).The Information from MSIS included age, sex, admission to hospital or nursing home, place of acquisition and if part of a known outbreak (from the Norwegian outbreak rapid alert system Vesuv) [9].The data obtained from the LIMS included sample date, sampling site/type of sample and laboratory results.The study group had access to identifiable information after data collection for the purpose of merging the two data sets correctly.All MRSA cases were categorized as carriage, infection, invasive infection or unknown based on sampling site/type of sample.Age groups were based on categories defined by Diaz et al. [10].Due to lack of temporal data for hospitalized patients and nursing home stays, a broad definition of healthcare-associated MRSA (HA-MRSA) was used.Accordingly, a case was categorized as HA-MRSA if the diagnosis occurred during a hospital or nursing home stay, or if MRSA was detected in healthcare workers (HCWs).Conversely, community-acquired MRSA (CA-MRSA) encompassed all other cases not falling within the HA-MRSA classification.

Bacterial strains, PCR and spa-typing
Bacterial strains were cultured on blood agar at 35˚C, after which extraction of DNA was performed by heat lysis.Briefly, a few colonies were suspended in molecular grade water and heated to 95˚C for 15 minutes with shaking (300 rpm).After centrifugation at 14,500 rpm for 2 minutes, the supernatant was collected.Confirmation of MRSA was performed with a multiplex conventional PCR detecting the mecA gene, the S. aureus-specific spa gene, and the Panton-Valentine leukocidin (PVL) genes lukSF-PV, followed by gel electrophoresis [11].For strains that were mecA negative, mecC PCR [5] was additionally performed.
All strains were spa-typed according to Harmsen et al. [11] with primers spa-1113f and spa-1514r [12] using the Ridom StaphType software and SpaServer [13].The spa-types were assigned to known sequence types (ST) and/or clonal complexes (CC) based on Ridom Staphtyper [12] and pubMLST [14,15] databases.If a spa-type could not be assigned to a ST or CC, multi locus sequence typing (MLST) was performed, and CC was assigned using eBURST [16] software.A minimum spanning tree (MST) based on spa-repeats was constructed using Based Upon Repeat Pattern (BURP) clustering [17] with the Ridom SeqSphere + software [18].To calculate genotypic diversity, Simpson's diversity index [19] was used.

Statistical analyses
Fisher's exact test was used for testing associations between the most frequent spa-types (�50 cases) and epidemiological variables (infection, carriage, HA, CA, outbreak, sex and age < 1).Binary logistic regression was used for testing interactions between age and sex, and the Chi-Square test (crosstable analysis) was used for testing for sex differences within age groups.Statistical analyses were performed using R studio v1.4 and IBM SPSS Statistics v29.0.0.0.The Benjamini-Hochberg method was used to correct for multiple hypothesis testing, with adjusted p-values < 0.05 regarded as statistically significant.

Results
In the 10-year study period a total of 15,200 MRSA cases were reported in Norway, from 14,386 patients (Table 1).The majority of patients had a single case of MRSA (95%), whereas 4% had two cases, and 0.6% of patients had three or more cases of MRSA.Among patients with multiple cases, 609 (87%) had the same spa-type, 84 (12%) had a different spa-type within the same CC and only 49 (7%) had a different spa-type of a different CC.Most commonly, the detection of multiple MRSA cases in a patient occurred in two consecutive years (61%), while 181 patients (26%) had an interval of 3-4 years between detection and 88 patients (12%) had an interval of � 5 years between detections.From 2008 to 2017, an increasing trend in notification rates of MRSA was observed, with an annual increase of 15% per 100,000 population.The notification rate rose from 14.2 in 2008 to 48.6 in 2017, resulting in an overall increase of 243% (Fig 1A).The primary driver of this increase was MRSA carriage, which accounted for 75% of the overall rise.In contrast, the rate of infections contributed to 22% of the overall increase.Notably, the incidence of invasive infections remained stable throughout the observed period, consistently below 0.5 per 100,000 population.
Of the MRSA cases that were confirmed with mecA/mecC PCR, the majority were positive for the mecA gene (99.9%),while only 16 cases (0-3 per year) were positive for the mecC gene (0.1%) (Table 2).For the remaining cases, there was no strain available for mecA/mecC PCR (5%).The proportion of MRSA cases that were positive for the toxin and epidemiological marker PVL ranged from 45-32%, showing a decreasing trend since a peak in 2011 (Table 2).The PVL positive proportion was significantly higher (p < 0.00001) for MRSA infections (59%) than for MRSA carriage (24%).
Of all MRSA cases, 30% had an epidemiological link to the healthcare system at the time of sampling, and were thus defined as HA-MRSA (Table 1).Of these, a majority (20%) were admitted to hospital, while 6% were healthcare personnel and 4% were nursing home patients.The 70% of remaining cases were defined as CA-MRSA.Throughout the study period, the incidence of HA-MRSA showed an increasing trend, from 5.2 per 100 000 in 2008 to 13.6 in 2017, of which MRSA in patients admitted to hospital was the major contributor (Fig 1B).CA-MRSA however showed a drastic increase, from 9.0 per 100 000 in 2008 to 35.0 in 2017.Some spa-types were statistically overrepresented within healthcare-associated MRSA, including MRSA t032 (60% HA), MRSA t065 (76%) and MRSA t067 (57%) (Table 3).Of these, t032 was found in all HA categories, whereas t067 was detected mainly in hospital admitted and nursing home patients and t065 was found mainly in nursing home patients.Notably, these genotypes were detected in older patient populations (median age � 70 years).
A total of 299 outbreak-related MRSA cases (2%) belonging to 53 different outbreaks were reported overall (Table 1), with a median outbreak size of only 4 cases (range 1-31 cases).The number of outbreaks and outbreak-related cases decreased markedly from the start of the period (2008-2011) to later in the period (2012-2017), with HA-MRSA and in particular nursing home-related outbreaks contributing to this decline (Fig 4).There was a large heterogeneity of genotypes associated to outbreaks, with a total of 38 different spa-types detected.The most frequent spa-types which were significantly associated with outbreaks were t304 (6% outbreak-associated), t032 (11%) and t688 (5%) (Table 3).Several of these genotypes were detected in older patient populations (median age � 70 years).Two MRSA spa-types were significantly associated with the age group neonates and infants (< 1 year), including t127 and t688 (Table 3).Of all MRSA cases in the study period, 4199 (28%) were registered as acquired in Norway and 3878 (26%) as acquired abroad, while the remaining cases (47%) had no information on place of acquisition (Table 1).Of the cases that were acquired abroad, Asia (48%) and Europe (23%) accounted for the largest groups, with the Philippines (10%), Syria (9%) and Spain (6%) being the main countries (Fig 5).The spa-types which were significantly associated to a specific country included MRSA t019 from the Philippines (46% associated with Philippines), t127 from Romania (54%), t002 from Sri Lanka (50%), t223 and t304 from Syria (28 and 16% accordingly) and t008 from USA (46%) (Table 4).

Discussion
Norway is considered a low-prevalence country in terms of MRSA incidence.However, we observed a substantial increase in the MRSA incidence from 2008 to 2017.This is in line with what has been reported for several of the Nordic countries [7].Although this likely reflects a real increase in MRSA cases in Norway, variation in screening practices has possibly also contributed.The majority of the increase in MRSA in the study period was attributed to community-associated cases and carriage, indicating that transmission is mainly occurring in the community.The incidence of MRSA infections was however also increasing, which may be a consequence of the substantial increase in carriage.Notably, the incidence of invasive infections remained relatively constant, below 0.5 per 100,000 population.
The incidence of HA-MRSA increased in the study period, primarily among patients admitted to hospitals.Due to lack of information on admission time, we used a broad definition of HA-MRSA, which might lead to overestimation.Furthermore, in line with the major increase of CA-MRSA in the same period, it is likely that part of the increase in HA-MRSA reflects spillover from the community.A declining trend was however observed for outbreakrelated cases, especially in nursing homes.The number of cases related to outbreaks are generally underestimated, especially for small and CA-associated outbreaks, which are less likely to be suspected and thus reported to the Norwegian rapid alert system.Nevertheless, it is possible that increased compliance with infection control measures in health-care facilities and Norway's "search and destroy" policy are contributing factors to the observed reduction.Different MRSA spa-types were significantly associated to either the healthcare setting or the community setting.In general, the spa-types associated with HA-MRSA had a patient population with higher median age (35-84 years) than the spa-types associated with CA-MRSA (13-32 years), likely reflecting the differences in setting where these clones were mainly spreading.An example of this is MRSA t032, a well-known HA-MRSA clone common in European countries [13], with a patient population of median age of 71, which was associated both to the healthcare setting and to outbreaks.Of the community associated MRSA spa-types, t223 was linked to acquisition from Afghanistan, Egypt, Russia and Syria, and found in a much younger population (median 23 years), likely due to the typically young age of immigrants from these countries.
The MRSA population in Norway was very heterogeneous, as neither of the major spatypes detected constituted more than 10% of the total MRSA cases, and most spa-types detected (90%) were found less than 10 times per year.These findings indicate that there are few successful clones and limited spread, either in the community or in the healthcare system.A previous study has indeed suggested that spread of MRSA in Norway is self-limiting (reproductive number of 0.68), and that the observed increase in incidence is mainly due to acquisitions from abroad [20].Based on the data on acquisition of MRSA in this study, 28% of the cases were acquired in Norway, while 26% were acquired abroad.However, this is often registered based on assumption, and for the majority of cases we had no information on place of acquisition.Thus, it is likely that these numbers are underestimated.There have been major changes in immigration-patterns to Norway during the 10-yearperiod [21], where the total number of immigrants per year increased from 2008-2011, and decreased from 2011-2017, mainly from Eastern European countries.From 2015-2017 there was a significant increase of immigration from Asia (especially Syria), related to the civil war in Syria.Other countries with large immigrant groups to Norway in the study period include Romania, Somalia and the Philippines.These patterns are to some extent reflected in our findings, where we observe relatively high numbers of spa-types associated to specific countries with high immigration to Norway in the period, including MRSA t127 (Romania), t019 (Philippines), t002 (Sri Lanka), t223 and t304 (Syria) and t008 (USA).
Several studies have previously shown that bacterial infections are more frequent in males than in females [22], and this was also reflected in our study.The causes are described to be multifactorial, stemming from genetic, anatomical, immunological, hormonal and behavioural differences between the sexes [23,24].Notably, we observed a significantly greater number of infections in males within the young adult (20-24 years) and adult (25-59 years) age groups, while females exhibited a higher prevalence of carriage strains.A possible contributing factor to this is that these age groups coincide with the prime childbearing years of women, potentially leading to more MRSA testing due to increased interaction with the healthcare system.Another important finding was that in the young adolescents group (10-14 years) we observed significantly higher rate of infections in females than in males.To our knowledge, this has not been previously reported, and could be attributed to using age groups stratified by life stage in this study.Although the reasons behind this finding are unclear, contributing factors may include onset of puberty and modulation of sex hormones on the immune system [23,25].
We observed that several MRSA spa-types were significantly associated either with infection (t002, t021, t044, t121 and t657), or with carriage (t386, t015, t304 and t127).Although there may indeed be biological differences in virulence traits that render some MRSA clones more virulent, this is outside of the scope of this work.The MRSA spa-types associated with infection did however not appear to be associated with higher age groups, and included both HAand CA-MRSA.Among them were some of the most common spa-types in Norway, the globally disseminated MRSA clones t002 and t008, as well as the multidrug resistant Bengal Bay clone t657 [26] and the European CA-MRSA clone t044 [7].No spa-types were significantly associated with invasive infection.However, in this study only 108 invasive strains were included, and this number was likely too low for sufficient statistical power.Nevertheless, t002, t019 and t008 were the most frequent MRSA spa-types causing invasive infections, and were also among the most frequent spa-types in Norway.A characteristic of the invasive infection group was that the patient population were mostly older adults (�60 years).Thus, invasive infection appears to be linked to high age, and likely with increasing comorbidities in this group.
Among the spa-types associated with carriage were MRSA t304 (CC6) and t127.Both of these have been considered CA-MRSA clones, and have also previously been linked to Middle Eastern countries [27,28] and South-Eastern Europe [29], respectively.The patient population of these spa-types were generally young, and thus presumably mostly healthy.However, if many become persistent carriers, it could possibly result in more infections in an older population.Furthermore, it is concerning that these MRSA spa-types have been reported to cause relatively large neonatal outbreaks, in Norway (unpublished data) and in Denmark [27,30].MRSA t127, along with MRSA t688, was significantly associated with neonates and infants (< 1 year) in this study, the latter also to outbreaks, and has previously been found in maternity wards in Sweden [31], Ireland [32] and Kuwait [33].

Conclusions
From 2008 to 2017, there has been a notable rise in the incidence of MRSA in Norway.This increase can be primarily attributed to CA-MRSA and carriage, while the incidence of infections and invasive infections have remained relatively low.Overall, there were significantly more MRSA infections in males than females.Interestingly, there was a significantly higher prevalence of MRSA infections in female young adolescents compared to males.The incidence of HA-MRSA has also increased, although it is still below 10 per 100,000 population.This suggests that the Norwegian "search and destroy" policy has been effective in preventing MRSA from becoming endemic in Norwegian healthcare institutions.Global patterns of travel, migration and work have however had large impact on the MRSA epidemiology in Norway, highlighting the importance of national and international surveillance in order to monitor temporal trends and spread of successful clones.Increased use of whole genome sequencing as well as more timely access to epidemiological data will hopefully enable an even more detailed view on the transmission of MRSA as well as a more rapid response to potential outbreaks in the future.

Fig 2 .
Fig 2. Minimum spanning tree with log distances based on BURP alignments of spa-repeats of MRSA cases in Norway 2008-2017.Only spa-types with more than five repeats were included (n = 14,140).Nodes are sized according to number of cases, and coloured according to assigned clonal complex as shown in the legend.https://doi.org/10.1371/journal.pone.0297333.g002

Table 1 . Clinical and epidemiological information on all MRSA cases in Norway 2008-2017. Number
(n) of cases and percentages (%) are given, with decimal values (> 2) rounded to the nearest whole number.

Table 2 . Temporal trends in molecular epidemiology of MRSA cases in Norway in 2008-2017.
The table shows frequency of mecA, mecC, PVL (infection and carriage) for all MRSA cases confirmed with PCR (n = 14,486).Decimal values (> 2) were rounded to the nearest whole number.

Table 3 . spa-types and associations to clinical and epidemiological data for MRSA cases in Norway in 2008-2017.
Number (n) of cases and percentages (%) are given, with decimal values (> 2) rounded to the nearest whole number.Median ages are shown for each spa-type within a clinical or epidemiological group.P-values reflect the statistical association (Fisher's exact test) of a clinical or epidemiological variable (first column) to a spa-type, with Benjamini-Hochberg-corrected p-values < 0.05 regarded as significant.

Table 4 . spa-types and associations to country for MRSA cases in Norway in 2008-2017.
Number (n) of cases and percentages (%) are given, with decimal values (> 2) rounded to the nearest whole number.P-values reflect the statistical association (Fisher's exact test) of a country (first column) to a spa-type, with Benjamini-Hochbergcorrected p-values < 0.05 regarded as significant. https://doi.org/10.1371/journal.pone.0297333.t004